ES2994737T3 - Polymerase chain reaction primers and probes for mycobacterium tuberculosis - Google Patents

Abstract

The present invention relates to novel molecular beacon and molecular beacon primers and probes for amplifying segments of different genes in Mycobacterium tuberculosis to identify the presence of M.tb DNA and/or resistance to anti-tuberculosis drugs. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Polymerase chain reaction primers and probes for Mycobacterium tuberculosis

Cross reference to related requests

The application claims priority from U.S. Provisional Patent Application No. 62/062,351, filed Oct. 10, 2014.

Government interests

The invention disclosed herein was made, at least in part, with government support under Grants Nos. U01AI082174 and R01AI080653 from the National Institutes of Health. Accordingly, the U.S. Government has certain rights in this invention.

Field of invention

This invention relates to novel sloppy molecular beacon (SMB) and molecular beacon (MB) primers and probes for amplifying and detecting segments of different genes in Mycobacterium tuberculosis (M.tb) in order to identify the presence of M.tb DNA and to identify resistance to anti-tuberculosis drugs.

Background of the invention

Tuberculosis (TB) was declared a global public emergency almost twenty years ago (WHO Global Tuberculosis Report 2013). Although the rate of new TB cases has been declining worldwide, the Millennium Development Goal of a 50% reduction in the disease by 2015 is unlikely to be met (WHO Global Tuberculosis Report 2013). An increase in the incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB is a serious threat to these reduction targets (WHO Global Tuberculosis Report 2013). MDR TB is defined as TB resistant to treatment with at least rifampicin and isoniazid and XDR TB is defined as MDR TB that is additionally resistant to treatment with the fluoroquinolone class of antibiotics and the injectable drugs amikacin, kanamycin and capreomycin. It is best to identify patients with drug-resistant TB as early as possible so that appropriate infection control and treatments can be initiated promptly Boehme, C. C., et al, 2011, Lancet 377:1495-1505.

Conventional phenotypic methods may take weeks to months to fully define the drug resistance pattern of Mycobacterium tuberculosis (Mtb) because of the very slow growth of this bacterium (Heifets, L., et al., J Clin Microbiol 38:1227-1230; Kim, S. J., 2005, Eur Respir J 25:564-569; and P. T., K., and K. G. P., 1985, Public health Mycobacteriology: A guide for level III laboratory. Center for Disease Control, US Department of Health and Human Services, Atlanta, Georgia.). Molecular assays offer the promise of more rapid detection of drug resistance. Mtb does not naturally contain drug resistance plasmids; therefore, molecular assays are directed against chromosomal DNA. Genotypic assays are relatively easy to design because the Mtb genome has a very high degree of sequence conservation. Virtually all drug-susceptible clinical isolates of Mtb have identical DNA sequences at drug resistance targets, except for a few easily identified “natural polymorphisms.” It follows that any deviation from the wild-type sequence in a drug resistance target gene indicates the presence of drug resistance to the corresponding drug. Genotypic assays are faster and more sensitive than phenotypic assays because DNA targets can be amplified by PCR. Biological hazards can be minimized by early killing of infectious organisms.

The genetic targets that account for the majority of drug resistance cases in TB are now well established. Real-time PCR remains the most sensitive, rapid and robust method for detecting mutations in bacteria. Virtually all other mutation detection methods, including PCR-MS, microarrays, miniarrays and next-generation sequencing, require nucleic acid amplification as a first step in the detection process. In contrast, real-time PCR allows amplification, detection and sample analysis to be performed in a single well. Tubes do not have to be opened, no complex fluidics are required. However, no one has been able to develop a broad drug resistance assay methodology that is simple and robust enough to be performed outside of reference laboratories. Therefore, there is a need for new primers and probes for the detection of M.tb and drug resistance of M.tb to the most commonly used first- and second-line drugs.

Summary of the invention

This invention relates to primers, probes and related uses in the detection of M.tb and drug resistance of M.tb.

In one aspect, the invention provides a kit as defined in claim 1.

In a second aspect, the invention provides a method as defined in claim 10.

In a third aspect, the invention provides a method as defined in claim 11.

Also disclosed is an isolated oligonucleotide set or primer set for amplifying a portion of a region of M. tuberculosis selected from the group consisting of genrpoB, gengyrA, gengyrB, deinhA promoter, genrrs, deeis promoter, genembB, genkatG, gendosR, gene IS6110, gene IS 1081. The set includes a pair of forward and reverse primers specific for the portion, wherein each primer has a sequence that is substantially identical to an oligonucleotide sequence selected from those listed in Tables 1A and 1B below. Accordingly, each primer has a sequence that is substantially complementary to the complement of the oligonucleotide sequence selected from those listed in the tables. In some embodiments, the primer sequence is identical to the oligonucleotide sequence selected from those listed in Tables 1A and 1B.

Also described is an isolated nucleic acid having a sequence that is substantially identical to one selected from those listed in Table 2. In some embodiments, the nucleic acid includes the sequence of one selected from those listed in Table 2. The nucleic acid can be labeled with, for example, a fluorophore and a quencher at its two ends respectively, or a fluorophore attached to an internal nucleotide in the probe. Examples of the fluorophores include fluorescein, cyanine 5, or TexasRed, and TAMRA. Examples of the quenchers include BHQ1, BHQ2, and DABCYL.

Also described is a kit containing one or more of the set of oligonucleotides and nucleic acid described above. The kit may further include a DNA polymerase, extension nucleotides, and a buffer.

Also described is a method for detecting drug resistance in M. tuberculosis. The method includes steps of amplifying a first nucleic acid target sequence with a first primer pair to generate a first amplicon, wherein (i) the first primer pair is specific for a portion of a region selected from the group consisting of genrpoB, gengyrA, gengyrB, deinhA promoter, genrrs, deeis promoter, genembB, and genkatGy and (ii) each primer has a sequence that is substantially identical to an oligonucleotide sequence selected from those listed in Tables 1A and 1B, and detecting a mutation in the first amplicon. The presence of the mutation is indicative of drug resistance. In the method, the detecting step may be carried out by various nucleic acid detection techniques known in the art, including, for example, sequencing-based techniques and techniques based on nucleic acid or peptide nucleic acid probe hybridization.

In one embodiment, the detecting step is performed by a process comprising (i) contacting the first amplicon with a first probe specific for the mutation under conditions conducive to hybridization to form a probe-target hybrid; (ii) performing a melting temperature (Tm) analysis to determine an assay Tm value for the probe-target hybrid; and (iii) comparing the assay Tm value to a predetermined reference Tm value. The assay Tm value, if different from the predetermined Tm value, indicates the presence of the mutation. For example, a shift in the assay Tm value of at least 3 (e.g., 3, 4, or 5) standard deviations from the reference Tm value indicates the presence of the mutation. Conversely, a shift in the assay Tm value of less than 3 standard deviations from the reference Tm value indicates the absence of the mutation. As disclosed herein, the predetermined reference Tm value may be the mean wild-type Tm value. In one example, the test Tm value, if less than the predetermined reference Tm value, e.g., by at least 3 standard deviations, indicates the presence of the mutation. Otherwise, the test Tm value, if not less than the predetermined reference Tm value, e.g., by 3 standard deviations, indicates the absence of the mutation.

The method may further include amplifying a second nucleic acid target sequence with a second primer pair to generate a second amplicon, the second primer pair being specific for a portion of a second region selected from the group consisting of genrpoB, gengyrA, gengyrB, deenhA promoter, genrrs, deeis promoter, genembB, and genkatG. In some embodiments, the first region is the genrrs or the deeis promoter. For example, the first region may be the genrrs and the second region may be the deeis promoter. The two regions may be amplified independently or amplified in the same reaction system using techniques such as nested PCR. In that case, the mutation may be an A1401G or C1402T mutation in the genrrs. The mutation may be within the deeis promoter region queried by the eis primer sequences.

The method described above allows the detection of resistance to a drug selected from the group consisting of isoniazid, rifampicin, amikacin, kanamycin, capreomycin, ethambutol and the fluoroquinolone class of drugs. The primer pair may be one selected from those listed in Tables 1A and 1B. The probe may have a sequence that is substantially identical or completely identical to one selected from those listed in Table 2.

Also described is a method for detecting the presence of M. tuberculosis in a test sample, e.g., from a subject. The method includes contacting the test sample with a first primer pair under conditions conducive to an amplification reaction to produce a first amplicon, and detecting the presence of the amplicon thereby detecting the presence of Mycobacterium tuberculosis in the test sample. The first primer pair may be a set of oligonucleotides for amplifying a portion of a region of M. tuberculosis selected from the group consisting of gengyrB, deinhA promoter, deeis promoter, genembB, genkatG, gendosR, gene IS6110, gene IS1081. Each primer of the first primer pair has a sequence that may be substantially identical to an oligonucleotide sequence selected from those listed in Table 1B. The method may further include contacting the test sample or the amplicon generated by the first primer pair with a second primer pair under conditions conducive to an amplification reaction to produce a second amplicon, and detecting the presence of the second amplicon. In that case, the presence of both the first amplicon and the second amplicon indicates the presence of Mycobacterium tuberculosis in the test sample.

Also described is a method for detecting the presence of M. tuberculosis in a test sample. The method includes contacting the test sample with a first molecular beacon probe under conditions conducive to a hybridization reaction to produce a probe-target hybrid, and detecting the presence of the probe-target hybrid thereby detecting the presence of Mycobacterium tuberculosis in the test sample. In this method, the first molecular beacon probe has a sequence that is substantially identical to one selected from those listed in Table 2. In one example, the first molecular beacon probe is selected from the group consisting of probe IS 1081, dosR2, and IS6110 (SEQ ID No.67-69).

Details of one or more embodiments are set forth in the description below. Other features, objectives, and advantages will become apparent from the description and claims.

Brief description of the drawings

Figure 1 is a diagram showing the detection of resistance to AMK and/or KAN in 603 clinical DNA samples using the three-point Tm profile generated by the SMB probe. Each of the three test SMBs was tested against all of the M. tb DNA samples in a multiplex PCR reaction. The results for each sample are shown as a three-point Tm plot on the X-axis, with the Tm value of each SMB indicated on the Y-axis. Isolates are ranked from left to right as phenotypically susceptible and then resistant. Distinct Tm shifts of at least one of the three probes can be observed for each resistant isolate. Figures 2A, 2B, and 2C are diagrams showing first derivatives of melting peak profiles of three SMB probes. Melting peak profiles of wild-type, mutant, and mixed DNA samples are shown for the SMBrrs-1400 SMB probe (2A), SMBeis1 probe (2B), and SMBeis2 probe (2C). Each melting curve represents an individual strain. Figure 3 is a diagram showing the MIC values of the derrsyeismutant and wild-type strains. Average MIC values for AMK and KAN of the derrsyeismutant and wild-type strains are shown. Error bars represent ± one standard deviation of the MIC values.eis-P indicates gene promoter.

Detailed description of the invention

This invention is based, at least in part, on an unexpected discovery of novel primers, SMB probes, and MB probes for amplifying segments of eleven different genes in M. tb to identify the presence of M. tb DNA and resistance to anti-tuberculosis drugs such as isoniazid, rifampin, amikacin, kanamycin, capreomycin, ethambutol, and the fluoroquinolone class of drugs.

Primers and probes

The primers described here that amplify the genes elrpoB, gyrA, gyrB, inhA promoter region, rrs, deis promoter region, embB, and katG allow a sensitive amplification of drug resistance-inducing mutation hotspots in M. tb. The corresponding SMB probes target and identify these mutations that result in drug resistance to the most commonly used first- and second-line drugs. These primers can be used with very high efficiency in both symmetric and asymmetric PCR assays. The primer and probe sequences described herein have been used by the inventors to develop rapid and accurate molecular drug susceptibility testing assays for M. tb. Aside from their obvious utility in molecular diagnostic assay for M. tb, these primers will also find use in sequencing the target genes to identify resistance-inducing mutations in surveillance assays and any other probe-based assays aiming at specific and sensitive identification of common drug resistance-inducing mutations in M. tb. The primer sequences amplifying the dosR, IS6110 and IS1081 genes enable highly sensitive and specific identification of M. tb and can be used in any PCR assay format intended for highly specific and sensitive molecular diagnosis of tuberculosis. Exemplary primers and probes of this disclosure are listed in the tables below.

Table 1A

Table 1B

Table 2

One or more of the probe sequences in Table 2 can be prepared in various detection formats, such as dual-labeled probes including linear probes, Taqman probes, molecular beacon probes, and sloppy molecular beacon probes. A "sloppy" probe refers to a probe that is mismatch tolerant. Mismatch-tolerant probes hybridize to and generate a detectable signal for more than one target sequence at a detection temperature in an assay, and various hybrids so formed will have different melting points. Single-stranded linear or random coil probes are generally mismatch tolerant. Examples of such probes are hairpin or linear probes with an internal fluorescent moiety whose fluorescence level increases upon hybridization to one or the other target strand. See, for example, Pat. US No.7662550 and 5925517, US 20130095479.

Preferably, the neglected probes are doubly labeled hairpin probes or molecular beacon probes, described in U.S. Pat. Nos. 7,662,550 and 5,925,517. These hairpin probes contain a target binding sequence flanked by a pair of arms complementary to each other. They may be DNA, RNA, or PNA, or a combination of all three nucleic acids. In addition, they may contain modified nucleotides and modified internucleotide linkages. They may have a first fluorophore on one arm and a second fluorophore on the other arm, wherein the absorption spectrum of the second fluorophore substantially overlaps the emission spectrum of the first fluorophore. Most preferably, such hairpin probes are "molecular beacon probes" having a fluorophore on one arm and a quencher on the other arm such that the probes are dark when free in solution. They may also be wavelength-shifting molecular beacon probes with, for example, multiple fluorophores on one arm that interact via fluorescence resonance energy transfer (FRET) and a quencher on the other arm. The target binding sequences may be, for example, 12 to 50 or 25 to 50 nucleotides in length, and the hybridization arms may be 4 to 10 or 4 to 6 (e.g., 5 or 6) nucleotides in length. Molecular beacon probes may be attached to primers, as described in U.S. Pat. Nos. 7662550 and 5925517 and WO 01/31062.

Neglected molecular beacon probes therefore refer to such a class of fluorescently labeled hairpin oligonucleotide hybridization probes. Such probes produce a detectable signal in a homogeneous assay, i.e. without having to separate probes hybridized to target probes from unbound ones. By virtue of their ability to bind to more than one variant of a given target sequence, the probes can be used in assays to detect the presence of a variant of a nucleic acid sequence segment of interest from among several possible variants or even to detect the presence of two or more variants. The probes can therefore be used in combinations of two or more in the same assay. Because they differ in the target binding sequence, their relative avidities for different variants are different. For example, a first probe may bind strongly to a wild-type sequence, moderately to a first allele, weakly to a second allele and not at all to a third allele; while a second probe may bind weakly to the wild-type sequence and the first variant, and moderately to the second variant and the third variant. Additional neglected probes will exhibit even different binding patterns due to their different target binding sequences. Thus, fluorescence emission spectra of neglected probe combinations define different microbial strains or species as well as allelic variants/gene mutation.

Since sloppy probes reproducibly fluoresce with varying intensities upon binding to different DNA sequences, combinations can be used in, for example, simple, rapid and sensitive nucleic acid amplification reaction assays (e.g. PCR-based assays) that identify multiple pathogens or variants in a single reaction vessel. It is understood, however, that the assays can also be performed on samples suspected of containing directly detectable amounts of unamplified target nucleic acids. This identification assay relies on analyzing the spectra of a set of partially hybridizing sloppy signaling probes, such as sloppy molecular beacon probes, each labeled with a fluorophore emitting light with a different wavelength optimum, to generate "signature spectra" of species-specific or variant-specific DNA sequences.

Using probes, multiplexing can be achieved, for example, by designing a different allele-discriminating molecular beacon probe for each target and differentially labeling each probe. (See, for example, US Pat. Nos. 7662550 and 5925517, WO 01/31062 and Tyagi et al. (2000) Nature Biotechnology 18: 1191-1196). Mixtures of allele-discriminating probes, each comprising aliquots of multiple colors, expand the number of probe signatures. To that end, each molecular beacon-target hybrid with a unique melting temperature will have the unique signal intensity corresponding to a defined probe and amplicon temperature and concentration. Thus, a limited number of neglected probes could be used as probes to identify many different possible target sequences in one real-time PCR reaction. Probes may be added to the amplification reaction mixture before, during, or after amplification. See US Patent No. 7,662,550.

Also described are kits containing reagents for performing the methods described above, including PCR and/or probe-target hybridization reactions. To this end, one or more of the reaction components, e.g., primers, polymerase, and PCR probes for the methods disclosed herein may be supplied in the form of a kit for use. In such a kit, an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate.

The kit also contains additional materials for practicing the methods described above. In some embodiments, the kit contains some or all of the reagents, materials for performing a method using primers and/or probes according to the disclosure. Some or all of the components of the kits may be provided in containers separate from or from the containers containing the primers and/or probes of the disclosure. Examples of additional components of the kits include, but are not limited to, one or more different polymerases, one or more control reagents (e.g., PCR primers or probes or control templates), and buffers for the reactions (in 1X or concentrated forms). The kit may also include one or more of the following components: supports, termination, modification or digestion reagents, osmolytes, and an apparatus for detection.

The reaction components used may be provided in a variety of forms. For example, the components (e.g., enzymes, probes, and/or primers) may be suspended in an aqueous solution or as a freeze-dried or lyophilized powder, pellet, or bead. In the latter case, the components, when reconstituted, form a complete mixture of components for use in an assay. The kits of the disclosure may be provided at any suitable temperature. For example, for storage of kits containing protein components (e.g., an enzyme) in a liquid, it is preferred that they be provided and maintained below 0°C, preferably at or below -20°C, or otherwise in a frozen state.

A kit or system of this disclosure may contain, in an amount sufficient for at least one assay, any combination of the components described herein. In some applications, one or more reaction components may be provided in pre-measured single-use quantities in individual, typically disposable tubes or equivalent containers. With such an arrangement, a PCR reaction may be performed by adding a target nucleic acid or a sample/cell containing the target nucleic acid to the individual tubes directly. The amount of a component supplied in the kit may be any appropriate amount, and may depend on the target market to which the product is directed. The container(s) in which the components are supplied may be any conventional container that is capable of containing the supplied form, for example, microcentrifuge tubes, ampoules, bottles, or integral assay devices, such as fluidic devices, cartridges, lateral flow, or other similar devices.

Kits may also include packaging materials for containing the vessel or combination of vessels. Typical packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, microparticles, and the like) containing the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like). Kits may further include instructions recorded in a tangible form for use of the components.

Definitions

A nucleic acid refers to a DNA molecule (e.g., but not limited to, a cDNA or genomic DNA), an RNA molecule (e.g., but not limited to, an mRNA), or a DNA or RNA analog. A DNA or RNA analog may be synthesized from nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded. An "isolated" nucleic acid is a nucleic acid, the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore encompasses, for example, (a) a DNA that has the sequence of part of a naturally occurring genomic DNA molecule, but is not flanked by both coding sequences flanking that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a PCR-produced fragment, or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein.

As used herein, the term "target nucleic acid" or "target" refers to a nucleic acid that contains a target nucleic acid sequence of interest. A target nucleic acid may be single-stranded or double-stranded, and is often double-stranded DNA. A "target nucleic acid sequence," "target sequence," or "target region" means a specific sequence that comprises all or part of the sequence of a single-stranded nucleic acid. A target sequence may be within a nucleic acid template or within the genome of a cell, which may be any form of single-stranded or double-stranded nucleic acid. A template may be a purified or isolated nucleic acid, or it may be unpurified or unisolated.

"Complementary" sequences, as used herein, may include, or be formed entirely from, Watson-Crick base pairs (e.g., A-T/U and C-G), non-Watson-Crick base pairs, and/or base pairs formed from non-natural and modified nucleotides, and insofar as the above requirements regarding their ability to hybridize are met. A complete complement or fully complementary may mean 100% (completely) or substantially complementary base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

"Substantially complementary" means that a nucleic acid or oligonucleotide has a sequence containing at least 10 contiguous bases that are at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to at least 10 contiguous bases in a target nucleic acid sequence such that the nucleic acid or oligonucleotide can hybridize or anneal to the target nucleic acid sequence under, for example, the hybridization condition of a PCR reaction or a probe-target hybridization condition. Complementarity between sequences can be expressed in a number of base mismatches in each set of at least 10 contiguous bases that are compared. The term "substantially identical" means that a first nucleic acid is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100%) complementary to a second nucleic acid such that the first nucleic acid is substantially complementary to and capable of hybridizing to the complement of the second nucleic acid under PCR hybridization or probe-target hybridization conditions. "Hybridization" or "hybridizing" or "hybridizing" or "pairing" refers to the ability of completely or partially complementary nucleic acid strands to come together under specified hybridization conditions in a parallel or preferably antiparallel orientation to form a stable double-stranded structure or region (sometimes referred to as a "hybrid" or "duplex" or "stem") wherein the two constituent strands are linked by hydrogen bonds. Although hydrogen bonds are typically formed between adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), other base pairs can form (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

A "nucleic acid duplex", "duplex", "stem", "nucleic acid hybrid" or "hybrid" refers to a stable nucleic acid structure comprising a hydrogen-bonded double-stranded region, for example, RNA:RNA, RNA:DNA, and DNA:DNA duplex molecules and analogs thereof. Such a structure can be detected by any known means, for example, by using a labeled probe, an optically active probe-coated substrate sensitive to mass changes on its surface (U.S. Pat. No. 6,060,237), or binding agents (U.S. Pat. No. 5,994,056).

As used herein, the term "amplification" and its variants includes any process for producing multiple copies or complements of at least some portion of a polynucleotide, with the polynucleotide typically being referred to as a "template." The template polynucleotide may be single-stranded or double-stranded. A template may be a purified or isolated nucleic acid, or it may be unpurified or unisolated. Amplification of a given template may result in the generation of a population of polynucleotide amplification products, collectively referred to as an "amplicon." The polynucleotides in the amplicon may be single-stranded or double-stranded, or a mixture of both. Typically, the template will include a target sequence, and the resulting amplicon will include polynucleotides having a sequence that is substantially identical to, or substantially complementary to, the target sequence. In some embodiments, the polynucleotides in a particular amplicon are substantially identical to, or substantially complementary to, each other; Alternatively, in some embodiments, the polynucleotides within a given amplicon may have nucleotide sequences that vary from one another. Amplification may proceed in a linear or exponential manner, and may involve repeated, consecutive repetitions of a given template to form two or more amplification products. Some typical amplification reactions involve successive, repeated cycles of template-based nucleic acid synthesis, resulting in the formation of a plurality of daughter polynucleotides that contain at least some portion of the nucleotide sequence of the template and that share at least some degree of nucleotide sequence identity (or complementarity) with the template. In some embodiments, each instance of nucleic acid synthesis, which may be referred to as an amplification "cycle," includes creating the free 3' end (e.g., by nicking a strand of a dsDNA) thereby generating a primer and primer extension steps; Optionally, an additional denaturation step may also be included where the template is partially or completely denatured. In some embodiments, a round of amplification includes a given number of repetitions of a single amplification cycle. For example, a round of amplification may include 5, 10, 15, 20, 25, 30, 35, 40, 50 or more repetitions of a particular cycle. In an exemplary embodiment, amplification includes any reaction where a particular polynucleotide template is subjected to two consecutive cycles of nucleic acid synthesis. Synthesis may include template-dependent nucleic acid synthesis.

Amplification may also include isothermal amplification. The term “isothermal” means carrying out a reaction at substantially constant temperature, i.e., without varying the reaction temperature at which a nucleic acid polymerization reaction occurs. Isothermal temperatures for isothermal amplification reactions depend on the strand-displacing nucleic acid polymerase used in the reactions. Generally, isothermal temperatures are below the melting temperature (Tm; the temperature at which half of the potentially double-stranded molecules in a mixture are in a single-stranded, denatured state) of the predominant reaction product, i.e., generally 90° C. or less, typically between about 20° C. and 75° C., and preferably between about 30° C. and 60° C., or more preferably at about 37° C.

The term "primer" or "primer oligonucleotide" refers to a nucleic acid strand or oligonucleotide capable of hybridizing to a template nucleic acid and that acts as a starting point for incorporating extension nucleotides depending on the composition of the template nucleic acid for nucleic acid synthesis. "Extension nucleotides" refers to any nucleotides (e.g., dNTPs) and analogs thereof capable of being incorporated into an extension product during amplification, i.e., DNA, RNA, or a DNA or RNA derivative, which may include a label.

As used herein, the term "oligonucleotide" refers to a short polynucleotide, typically less than or equal to 300 nucleotides in length (e.g., in the range of 5 to 150, preferably in the range of 10 to 100, more preferably in the range of 15 to 50 nucleotides in length). However, as used herein, the term is also intended to encompass longer or shorter polynucleotide chains. An "oligonucleotide" may hybridize to other polynucleotides, thereby serving as a probe for polynucleotide detection, or a primer for polynucleotide chain extension.

The term "probe" as used herein refers to an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, typically through complementary base pairing, typically through hydrogen bond formation. Probes may bind to target sequences that lack complete complementarity to the probe sequence depending on the stringency of the hybridization conditions. There may be any number of base pair mismatches that will interfere with hybridization between the target sequence and the single-stranded nucleic acids described herein. However, if the number of mutations is so large that hybridization cannot occur even under less stringent hybridization conditions, the sequence is not a complementary target sequence. A probe may be single-stranded or partially single-stranded and partially double-stranded. The stranding ability of the probe is dictated by the structure, composition, and properties of the target sequence. Probes can be labeled directly or indirectly labeled with a marker such as biotin to which a streptavidin complex can subsequently bind.

The term "detection probe" refers to an oligonucleotide having a sequence sufficiently complementary to its target sequence to form a stable probe:target hybrid for detection under stringent hybridization conditions. A probe is typically a synthetic oligomer that may include bases complementary to the sequence outside the target region that do not prevent hybridization under stringent hybridization conditions to the target nucleic acid. A sequence non-complementary to the target may be a homopolymer stretch (e.g., poly-A or poly-T), promoter sequence, restriction endonuclease recognition sequence, or sequence to confer the desired secondary or tertiary structure (e.g., a catalytic site or hairpin structure), or a tag region that can facilitate detection and/or amplification. "Stable" or "stable to detection" means that the temperature of a reaction mixture is at least 2°C below the melting temperature (Tm) of a nucleic acid duplex contained in the mixture, more preferably at least 5°C below the Tm, and even more preferably at least 10°C below the Tm.

A "label" or "reporter molecule" is a chemical or biochemical moiety useful for labeling a nucleic acid (including a single nucleotide), polynucleotide, oligonucleotide, or protein ligand, e.g., amino acid or antibody. Examples include fluorescent agents, chemiluminescent agents, chromogenic agents, quenching agents, radionucleotides, enzymes, substrates, cofactors, inhibitors, magnetic particles, and other moieties known in the art. Labels or reporter molecules are capable of generating a measurable signal and may be covalently or non-covalently linked to an oligonucleotide or nucleotide (e.g., a non-naturally occurring nucleotide) or ligand.

As used herein, the term "contacting" and its variants, when used in reference to any set of components, includes any process by which the components to be contacted are mixed in the same mixture (e.g., added into the same compartment or solution), and does not necessarily require actual physical contact between the stated components. The stated components may be contacted in any order or any combination (or subcombination), and may include situations where one or some of the stated components are subsequently removed from the mixture, optionally prior to the addition of other stated components. For example, "contacting A with B and C" includes any and all of the following situations: (i) A is mixed with C, then B is added to the mixture; (ii) A and B are mixed into a mixture; B is removed from the mixture, and then C is added to the mixture; and (iii) A is added to a mixture of B and C. “Contacting” a target nucleic acid or a cell with one or more reaction components, such as a polymerase, primer set, or probe, includes any or all of the following: (i) the target or cell is contacted with a first component of a reaction mixture to create a mixture; other components of the reaction mixture are then added in any order or combination to the mixture; and (ii) the reaction mixture is completely formed prior to mixing with the target or cell.

The term "mixture" as used herein refers to a combination of elements, which are interspersed and not in any particular order. A mixture is heterogeneous and not spatially separable into its different constituents. Examples of mixtures of elements include several different elements that are dissolved in the same aqueous solution, or several different elements attached to a solid support randomly or in no particular order where the different elements are not spatially distinct. In other words, a mixture is not addressable.

As used herein, the term "subject" refers to any organism having a genome, preferably, a living animal, e.g., a mammal, that has been the subject of diagnosis, treatment, observation, or experiment. Examples of a subject may be a human, a livestock animal (cattle and dairy cattle, sheep, poultry, pigs, etc.), or a companion animal (dogs, cats, horses, etc.).

A "sample" as used herein means any biological fluid or tissue obtained from an organism (e.g., patient) or from components (e.g., blood) of an organism. The sample may be any biological tissue, cell(s), or fluid. The sample may be a "clinical sample" which is a sample derived from a subject, such as a human patient or a veterinary subject. Useful biological samples include, without limitation, whole blood, saliva, urine, synovial fluid, bone marrow, cerebrospinal fluid, vaginal mucus, cervical mucus, nasal secretions, sputum, semen, amniotic fluid, bronchoalveolar lavage fluid, and other cellular exudates from a patient or subject. Such samples may be further diluted with saline, buffer, or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means. Biological samples may also include tissue sections such as frozen sections taken for histological purposes. A biological sample may also be referred to as a "patient sample." A biological sample may also include a substantially purified or isolated protein, membrane preparation, or cell culture.

The terms "determine", "measure", "evaluate" and "assess" are often used interchangeably and include both quantitative and qualitative measurement, and include determining whether a characteristic, trait or quality is present or not. Assessment may be relative or absolute. Assessing the presence of a target includes determining the amount of the target present as well as determining whether it is present or absent.

As used herein, the term "reference" value refers to a value that statistically correlates with a particular outcome as compared to an assay result. In preferred embodiments, the reference value may be determined from a statistical analysis examining the mean of the wild-type values. The reference value may be a threshold score value or a cutoff score value. Typically, a reference value will be a threshold above (or below) which one outcome is more likely and below which an alternative outcome is more likely.

As disclosed herein, a difference in values is indicative of the presence or absence of a pathogen (e.g., Mycobacterium tuberculosis) or a mutation. The phrase "difference" in level or value refers to differences in a variable (e.g., Tm) of an analyte (e.g., a probe-target hybrid) in a sample compared to a control or reference level or value. In one embodiment, a difference in value or level may be a statistically significant difference between the amounts of an analyte present in a sample compared to a control. For example, a difference may be statistically significant if the measured level of the analyte falls outside of about 1.0, 2.0, 3.0, 4.0, or 5.0 standard deviations of the mean of any control or reference group.

As disclosed herein, various ranges of values are provided. It is understood that each intermediate value, up to one-tenth of a unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intermediate value in a stated range and any other stated or intermediate value in that stated range is included within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range where one, neither, or both limits are included in the smaller ranges is also included within the disclosure, subject to any limits specifically excluded in the stated range. Where the stated range includes one or both of the limits, ranges that exclude one or both of those included limits are also included in the disclosure. The term "about" generally refers to plus or minus 10% of the stated number. For example, "about 10%" can indicate a range of 9% to 11%, and "about 20" can mean 18-22. Other meanings of "about" may be apparent from context, such as rounding, so, for example, "about 1" can also mean 0.5 to 1.4.

Uses

Recently, two separate SMB assays have been described that rapidly and reliably identify M. tb mutations that are largely responsible for rifampin and fluoroquinolone (FQ) resistance (Chakravorty, S., B. et al., 2011, J Clin Microbiol 49:932-940; Chakravorty, S., H., et al., 2012, J Clin Microbiol 50:2194-2202). The assays disclosed herein have the advantage of being in real-time PCR format, so they are easy to use and not subject to cross contamination by amplicons. Furthermore, mutation detection has been shown to be robust and amenable to high-throughput testing. The examples below present a TB SMB drug resistance detection assay system, adding assays that allow detection of AMK and KAN resistance. The causes of discordance between the assay disclosed herein and phenotypic susceptibility testing methods were also explored. The results show that some of the most commonly used phenotypic methods may fail to detect M. tb isolates with resistance-conferring mutations if these mutations only moderately increase the minimum inhibitory concentrations (MICs) for KAN. New mutations in whiB7 that are associated with low-level resistance to KAN were also discovered.

As disclosed herein, the multiplexed SMB PCR and melting assay accurately identified mutations in the dei gene and promoter associated with AMK and/or KAN resistance. The assay did not produce false resistance calls when tested against NTM, Gram-positive, and Gram-negative bacteria. The majority of instances of hetero-resistance were also detected by the assay, when present. Unlike the MTBDRsl platform, the assay can be performed in a closed real-time PCR system, and can be easily adapted to high-throughput testing as all assay steps are performed in 384-well plates. The SMB assay also avoids potential problems associated with alternative methods for mutation detection. High-resolution melting curve analysis requires the ability to detect subtle variations in melting curves (Yadav, R., S., et al., 2012, J Appl Microbiol 113:856-862). Other post-PCR melting-based molecular assays must detect mutations by decoding complex fluorescence contours (Rice, J. E., et al., 2012. Nucleic Acids Res 40:e164). In contrast, the SMB assay produces clear, easily distinguishable Tm peaks and definitive Tm shifts to identify mutations of interest. Individual Tm values can also be used to group samples that have the same genotype.

As disclosed herein, an assay was tested on a panel of 603 clinical specimens representing both new TB cases and unresolved retreatment cases, and the relationship of the target mutations to the susceptibility pattern of the clinical isolates was assessed. It was observed that 100% of the isolates with A1401G mutations were strongly correlated with high level resistance to both AMK and KAN. However, mutations in the promoter resulted in only moderate to low level resistance to KAN and no resistance to AMK, which is consistent with previous studies (Campbell, P. J., et al, 2011, Antimicrob Agents Chemother 55:2032-2041; Zaunbrecher, M. A., et al., 2009, Proc Natl Acad Sci U S A 106:20004-20009). The study also showed here that the LJ absolute concentration method for susceptibility testing does not adequately detect moderate to low level of KAN resistance. Indeed, almost two-thirds of the samples with mutations in the deeisse promoter were detected as susceptible to KAN on LJ medium. However, all but one sample with mutations in the deeisse promoter were detected as resistant to KAN by the MGIT method. Two such isolates contained a C(-12)T eneis mutation. These mutants were also resistant to KAN when tested by MYCOTB, which showed a KAN MIC of 5 μg/ml. Previous studies have suggested that clinical isolates with C(-12)T mutations do not correlate (Zaunbrecher (2009) or correlate poorly (Campbell, 2011; Hoshide, M., L. et al.,2014, J Clin Microbiol 52:1322-1329.) with KAN resistance. These studies possibly missed the relationship between this mutation and low level KAN resistance due to the assay method used to establish phenotypic susceptibility. These results suggest that the MGIT or MYCOTB methods should be preferred for assaying phenotypic resistance to KAN. They also highlight the power of genotypic resistance testing, such as that disclosed herein, to detect mutations that cause low level resistance and may be missed by phenotypic testing alone (Rigouts, L., M. et al.,2013, J Clin Microbiol 51:2641-2645; Sirgel, F. A., et al., 2012, Microb Drug Resist 18:193-197; and Van Deun, A., et al.,2013, J Clin Microbiol 51:2633-2640).

The study set here included a sample that was a mixture of wild-type and C1402T derrs mutants. This sample was susceptible to both AMK and KAN on LJ medium. Isolates with C1402T mutations have been reported to be susceptible to AMK, but resistant to KAN (Maus, C. E., et al., 2005, Antimicrob Agents Chemother 49:3192-3197). In this particular case, repeated susceptibility assays using LJ medium showed susceptibility to KAN presumably due to the hetero-resistant nature of the sample. Here, the molecular assay served as a better predictor of potentially emerging resistance than the phenotypic assay, as the SMB assay clearly detected the presence of both wild-type and mutant DNA types.

The incidence of mutations in 1484 derrs in clinical strains with resistance to AMK or KAN has been very low (Georghiou, S. B., et al., 2012, PLoS One 7: e33275) making their clinical significance debatable. A separate version of the assay that targeted the 1484 derrs codon did not detect any mutations in any of the 603 isolates in the study, as well as in an additional 259 isolates from the New Jersey-New York area, which included 33 isolates resistant to both AMK and KAN. The absence of any mutations in 1484 derrs in this expanded study set was confirmed by Sanger sequencing (data not shown). In light of the very low prevalence of mutations in 1484 derrs, this codon is unlikely to provide much value in predicting aminoglycoside resistance. Therefore, it is recommended that molecular assays for aminoglycoside resistance target only codons 1401-1402 in the rrs gene.

Twenty-two AMK- or KAN-resistant samples were found to have wild-type sequences in the gene rrs and promoter region of deis. A recent study has shown a possible association between mutations in the 5'UTR of whiB7 and KAN resistance, identifying a mutation in the 5'UTR of whiB7 in a single clinical strain with unexplained KAN resistance (Reeves, A. Z., et al., 2013, Antimicrob Agents Chemother 57:1857-1865). Several novel 5'UTR mutations of whiB7, as well as a deletion, were also described that appear to be associated with KAN resistance. No suitable universal biomarkers have been identified that can explain resistance to KAN and AMK in the remaining 15-20% of clinical strains with wild-type rrs,deis promoter region. Samples containing wild-type gene DNA and the promoter region mixed with a trace amount of mutant targets from a KAN- or AMK-resistant subpopulation could also explain the remaining discordances between the phenotypic resistance tests and the SMB assay disclosed herein. However, expensive investigation of hetero-resistance was beyond the scope of this study. Some recent studies have suggested that the PPE60 and Rv3168 genes might be involved in unexplained KAN resistance (Fahrat, M. R., et al.,2013. Nat Genet 45:1183-1189; Zhang, H., et al., 2013, Nat Genet 45:1255-1260) although this remains to be verified in clinical settings.

In summary, a sensitive and specific assay for the detection of AMK and KAN resistance in M.tb was developed and validated in clinical isolates with a high prevalence of MDR and XDR TB. The results show that A1401G mutations encode a high level of cross-resistance to both AMK and KAN, and that mutations in the dei promoter encode moderate to low level KAN resistance, which is consistent with previous functional genomic studies (Zaunbrecher 2009). Comparison of the performance of the assay disclosed herein with three different phenotypic susceptibility testing methods on solid and liquid media revealed that low to moderate level KAN resistance caused by mutations in the dei promoter is largely missed by LJ-based susceptibility testing. These results strongly argue for the value of genotypic testing for detecting aminoglycoside resistance, and the results demonstrate the specific utility of the SMB-based assay disclosed herein.

Examples

Example 1

This example describes materials and methods used in EXAMPLES 2-7 below.

DNA samples

The M. tb test samples consisted of DNA isolated from 603 sequential M. tb isolates cultured from 503 patients enrolled in a natural history study of MDR-TB (NCT00341601 at clinicaltrials.gov) at Masan National Hospital in Changwon, Republic of Korea. Two cohorts were tested. Cohort A consisted of treatment-naïve newly suspected TB cases (158 samples) and cohort B consisted of retreatment TB cases (445 samples). Fresh sputum samples were collected from each patient at the start of treatment and cultured for M.tb. In a subset of patients, repeat sputum samples were collected at the 1st, 4th, and 6th months of treatment and also cultured for M.tb. Non-tuberculous mycobacteria (NTM) and gram-positive and gram-negative bacteria test samples were taken from the New Jersey Medical School (NJMS) DNA repository as previously described (Chakravorty, S., 2012. J Clin Microbiol 50:2194-2202). Phenotypic drug susceptibility assay

Phenotypic drug susceptibility testing was performed on the 603 isolates by the absolute concentration method in LJ medium to determine susceptibility to AMK and KAN using breakpoint concentrations of 40 µg/ml (the standard concentration used during 2012 when the isolates were tested) for both antibiotics (Jnawali, H. N., 2013, Diagn Microbiol Infect Dis 76:187-196) at the International Tuberculosis Research Centre (ITRC), South Korea. MICs for AMK and KAN for 173/603 samples were also assessed using TREK Sensititre<®>MYCOTB MIC Plates ("MYCOTB"; TREK Diagnostic Systems, Cleveland, OH, USA) as previously described (Lee, J., 2014, Antimicrob Agents Chemother 58:11-18). For 560/603 samples, KAN resistance was also assessed using the Mycobacterial Growth Indicator Tube (MGIT) system (Becton Dickinson, Franklin Lakes, NJ, USA) at a critical concentration of 2.5 μg/ml. For samples with phenotypic susceptibility test results that were discordant with Sanger sequencing results of the target genes, phenotypic susceptibility testing was repeated to confirm the initial findings. In cases where MGIT and LJ susceptibility test results showed discordance, both assays were repeated to confirm or rectify the initial findings.

DNA preparation and sequencing

DNA for both SMB assay testing and Sanger sequencing was prepared from cultured isolates by boiling a loopful of culture in 200 μl of Instagene Matrix resin (Bio-Rad Laboratories, Hercules California, USA) in the presence of 0.1% Triton X100 for 10–15 min. The supernatant was recovered after centrifugation and quantified using a Nanodrop microvolume spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). For Sanger sequencing, two different fragments of the rrs gene (nucleotides 420-980 and 1293-1537) and a part of the 5’ deeisen coding sequence plus the entire deeisse promoter were amplified using 0.5 μM of forward and reverse primers, 1X PCR buffer, 250 mM dNTPs, 22.5 mM MgCl and 0.03 U/µl of AmpliTaq Gold DNA polymerase enzyme (Applied Biosystems, Foster City, CA, USA) according to the following parameters: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s, 58-60 °C for 30 s and 72 °C for 10-30 s depending on the amplicon size. The promoter region and whiB7 gene fragments were amplified as previously described (10, 33). For a subset of samples, a 538 bp fragment of the whiB7 gene including 412 bp of the 5' untranslated region and 126 bp of the ORF was amplified and sequenced using primers whiB7F 5'aaacgcgcaggtcagaaaat 3' and whiB7R 5'cagtgtcttggctacctcga 3' (SEQ ID No: 70 and 71). Additionally, a 275 bp fragment of the whiB7 gene, which included almost the complete whiB7 ORF, was also amplified using the primers whiB7-ingen-F 5' GTCGGTACTGACAGTCCCC 3' and whiB7-ingen-R 5'ATGCAACAGCATCCTTGCG 3' (SEQ ID No: 72 and 73). The PCR products were subjected to bidirectional sequencing using the gene-specific forward and reverse primers on a 3130XL Genetic Analyzer (Applied Bio-systems, Foster City, CA, USA) using a BigDye Terminator, version 3.1, cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions.

Molecular beacons and assay primers

SMB assays target mutations in M.tb at codons 1401 and 1402 of the gene and mutations along the promoter region of the gene. A 113 bp fragment (nucleotides 1335-1451) of the gene was amplified using primers AMG-F (5'-GCTAGTAATCGCAGATCAGCAACGCTGC-3', SEQ ID No: 51) and AMG-R (5'-CCTCCCGAGGGTTAGGCCACT-3', SEQ ID No: 52) and a 98 bp fragment encompassing the promoter region and the five initial codons of the gene (nucleotides -81 to 17) was amplified using primers eis-F (5'-CACAGGGTCACAGTCACAGAATC-3', SEQ ID No: 18) and eis-R (5'-GCATCGCGTGATCCTTTGCCAGAC-3', SEQ ID No: 53). Primers derrsse were designed to be specific for the genus Mycobacterium and primers deeisse were designed to be specific for the M.tb complex. One probe SMBrrs-1400 (5'-6-carboxyfluoresceincacgaccgcccgtcacgtcatgaaagtcggtcgtg-BHQ1-3', SEQ ID No: 59) and two probes SMBeis-1 (5'-Cyanine5-caggcggtcgtaatattcacgtgcacctggccgccgcctg-BHQ2-3', SEQ ID No: 16) and eis-2 (5'-TexasRedctgcggcatatgccacagtcggattctctgacgcgag-BHQ2-3', SEQ ID No: 61) (where the underlined sequences represent the stem portion of SMB and BHQ represents the black hole screener) are directed against the gene rrs and the promoter region of eis, respectively. The derrs probe was designed to be complementary to the antisense strand and the deeisse probes were designed to be complementary to the sense strand. SMBs were designed using the in silico DNA folding program at http://mfold.rna.albany.edu/?q_mfold/dna-folding-form, and the probe-target hybrid folding program at http://mfold.rna.albany.edu/?q_DINAMelt/Two-state-melting was used to predict possible probe-target hybrid structures and melting temperatures (Tm). Probes were designed to generate a maximal Tm difference between wild-type and mutant sequences in their respective target regions to allow unambiguous identification of mutations. Primers were obtained from Sigma Aldrich (St. Louis, Missouri, USA) and SMB probes from Biosearch Technologies (Novato, California, USA).

Test procedure

All samples were independently coded and randomized to ensure that assay validation was performed in a blinded manner. The assay was tested at both the ITRC in Masan, South Korea and at New Jersey Medical School (NJMS), Rutgers, Newark, New Jersey. After testing of the entire set of 603 samples was completed, the samples were decoded and PCR results were compared to corresponding sequencing and phenotypic drug susceptibility testing results. Results obtained at each site were also compared. Assay results were not reported to treating physicians and were not used to guide any treatment decisions. PCR was performed in 384-well plates using a Roche Light Cycler 480 II Real-Time PCR System (Roche Diagnostics Co. Indianapolis, Indiana, USA) in 20 µl reaction volumes containing 100 nM forward primer and 1 µM reverse primer for the gene, 1 µM forward primer and 50 nM reverse primer for the deeis promoter region, 1 ng/µl each of derrs-1400 and eis-1 probes and 0.8 ng/µl of eis-2 probes, 24 mM MgCl, 250 mM deoxynucleoside triphosphates (dNTPs), 1XPCR buffer, 8% glycerol, 0.06 U/µl Platinum<®>TfiExo(-) DNA polymerase (Life Sciences, Inc., Indianapolis, IN, USA). Technologies, Grand Island, NY, USA) and 2–5 ng of sample DNA or an equivalent volume of water. PCR was carried out with the following steps: enzyme activation for 2 min at 95 °C, followed by 50 cycles of denaturation at 95 °C for 10 s and combined annealing and extension at 67 °C for 30 s. After PCR cycling, post-PCR-Tm analysis was performed by denaturation at 95 °C for 2 min, followed by cooling to 45 °C and then gradual heating to 85 °C, with continuous fluorescence monitoring during the process at a rate of 1 data acquisition per degree Celsius. Tm values were identified at the end of the reaction using Tm calling software (Light Cycler 480 software). However, each Tm was also verified by a trained observer before the final Tm value identification was performed. Samples showing distinct double peaks for any probe corresponding to the wild-type and mutant Tm were considered indicative of hetero-resistance. A no-template control using sterile water instead of DNA as template was used as a negative DNA control, and a positive DNA control using 1 ng of M. tbH37Rv genomic DNA as template was also included in each assay plate.

Approval of human subjects

This study was approved by the Institutional Review Boards of Masan National Hospital, NIAID, and Rutgers (formerly UMDNJ), and all subjects provided written informed consent (Rutgers IRB protocol number 0120090104).

Example 2. Identification of Tm values associated with wild-type and mutant sequences

The SMB-based assay disclosed herein detected resistance to AMK and KAN by searching for mutations in the M.tb gene and promoter that have known associations with resistance. The assay consisted of a PCR step followed by Tm analysis in the presence of SMB probes complementary to portions of the M.tb target amplicons. The inventors first evaluated the ability of the assay to identify target mutations in artificial oligonucleotides and sequenced DNA templates from selected wild-type and mutant M.tb strains (data not shown). Wild-type sequences were identified by the presence of Tm values within 1 °C of the known mean values for the wild-type targets. Mutant sequences were identified by a shift in Tm values of at least five standard deviations from the mean wild-type Tm values. The ability of the assay to detect the most prevalent mutations associated with AMK and KAN resistance was then assessed in clinical DNA samples. Assays were performed on a panel of 603 clinical samples, consisting of 487 samples with wild-type sequences and 116 samples with mutations in the test targets. Five of these samples had mixtures of both wild-type and mutant DNA detected by Sanger sequencing. The SMB assay correctly identified 115/116 (99%) mutant or mixed samples (heterogeneous samples containing both mutant and wild-type DNA) as mutant or mixed and 487/487 (100%) pure wild-type samples as wild-type. A single mixed sample (as indicated by Sanger sequencing) was identified as a wild-type sample by the assay disclosed herein. The Tm values produced by each SMB probe in the background of wild-type or mutant targets were highly reproducible. For wild-type targets, rs-1400, eis-1, and eis-2 probes showed mean Tm values of 70.1 °C ± 0.15, 63.9 °C ± 0.19, and 69 °C ± 0.23, respectively (Table 3). For mutant Tm targets, A1401G and C1402T, the mutations resulted in a decrease of 3.9 °C (± 0.17) and 5.6 °C (± 0.21) in Tm values for rs-1400 probe, respectively (Table 3). Similarly, the eis-1 and eis-2 probes robustly detected a range of mutations in the eis promoter region as a mutant resulting in a 4.3°C to 6.5°C decrease in Tm values, compared to the expected wild-type Tm values (Table 3). PCR assays performed at the two different laboratories at Rutgers and ITRC were in complete agreement for all samples detected as wild-type and mutant, as well as mixtures.

The assay results allowed us to clearly segregate the 603 samples into wild-type and mutant Tm cluster types based on their individual three-spot Tm patterns (Fig. 1). The assay correctly identified mutations in all 75 samples containing only the A1401G mutation (Table 3, Fig. 2, panel A). Three of the four samples containing mixtures of A1401G and wild-type sequence were also detected as mixed wild-type/mutant based on the presence of a double Tm peak. A single sample containing a mixture of the C1402T mutation and wild-type DNA was also identified by the presence of double Tm peaks from the sample with a mutant-specific Tm for the C1402T mutation (Table 3, Fig. 2, panel A). The 32 samples with mutations in the deeis promoter region included five different polymorphisms (at positions -8, -10, -12, -14, and -37). All of these mutations were successfully detected by any one of the deeis SMBs (Table 3, Fig. 2, panels B and C). Four samples that had mutations in both the deeis gene and the deeis promoter region were also successfully detected as double mutants (Table 3). Sequencing of the deeis gene did not identify any sample with a codon 1484 mutation, regardless of its drug susceptibility pattern. Example 3. Identification of amikacin resistance

In this example, assays were performed to evaluate the performance of the molecular assay in relation to the results of phenotypic drug susceptibility testing. The apparent performance of a genotypic drug susceptibility test can vary depending on the mutations selected for inclusion in the test and the phenotypic assay used as the gold standard (Kim, S. J. 2005 Eur Respir J 25:564-569; Rigouts, L., et al., 2013, J Clin Microbiol 51:2641-2645; and Van Deun, A., et al.,2013, J Clin Microbiol 51:2633-2640). Considering the LJ-based drug susceptibility testing method as the gold standard (performed for all 603 study samples), the SMB derrs Tm, characteristic of the A1401G mutation, classified 82/90 of the AMK-resistant samples as resistant, (sensitivity 91.1%; 95% CI, 82.8% to 96.8%). The wild-type Tm classified 512/513 of the AMK-susceptible samples as susceptible. One individual isolate among the 513 AMK-susceptible isolates was identified as a mixture of wild-type and C1402T mutant DNA by the SMB assay disclosed herein due to the presence of a clear double peak generated by the SMB derrs probe, corresponding to the wild-type Tm and a C1402T mutant-specific Tm (Figure 2, panel A). This was also confirmed by Sanger sequencing. Since previous studies have shown that the C1402T mutation does not encode AMK resistance (24), the specific Tm corresponding to the C1402T mutation can be considered as an indicator of AMK susceptibility. This consideration resulted in the assay disclosed herein correctly detecting all 513/513 AMK susceptible samples, resulting in a specificity of 100% (95% CI, 99 to 100%). Inclusion of the characteristic Tm values for mutations in the deei promoter region in the analysis did not increase the sensitivity for detecting AMK resistance, but decreased the specificity from 100% to 93.8% (95% CI, 91.2 to 95.6%). These results are consistent with previous reports suggesting that mutations in the deeisno promoter are associated with AMK resistance as defined by the LJ drug susceptibility assay (Campbell 2011 and Zaunbrecher 2009).

Example 4. Identification of kanamycin resistance

The performance of an assay to detect KAN resistance was also evaluated using LJ-based drug susceptibility testing as a gold standard for all 603 samples. Using the Tm values generated by the herein-disclosed dersMB typical for the A1401G or C1402T mutation to define resistance, the assay detected 82/106 samples as KAN resistant (sensitivity 77.4%; 95% CI 68.0 to 84.7%). In contrast, using a dersMB Tm characteristic of the wild-type target to define susceptibility, it identified 496/497 KAN-susceptible samples as susceptible (Table 4) (specificity 99.8%; 95% CI 98.7 to 100%). The addition of Tm values from the two SMBs characteristic of mutations in the deiisse promoter region to the definition of resistance increased the sensitivity for detecting KAN resistance from 77.4% to 87.7% (95% CI, 79.5% to 93%), as 11 additional KAN-resistant samples were classified as resistant. However, specificity decreased from 99.8% to 95.6% (95% CI, 93.3% to 97.1%) as 21 KAN-susceptible samples with deiisse promoter mutations were now “falsely” detected as KAN-resistant (Table 4).

A similar analysis was then performed using MGIT-based drug susceptibility assay results as the gold standard for the 560 of the samples for which an MGIT result was available. This subset included all samples harboring only mutations in the deeis promoter. Comparison of the assay results with the MGIT-based gold standard helped to elucidate deeis mutants with discordant KAN resistance on LJ medium. Using MGIT as the gold standard and only the characteristic SMB Tm values for the A1401G or C1402T mutations to define KAN resistance, only 63/113 KAN-resistant samples were identified as resistant by the SMB assay (sensitivity 55.8%; 95% CI, 46.1 to 65%). Conversely, using the SMB Tm characteristic of the wild-type target to define susceptibility identified 445/447 samples as susceptible to KAN (specificity 99.8%; 95% CI, 98.5 to 100%; Table 4). In contrast to the case with LJ-based susceptibility testing, including the Tm values characteristic of the deei promoter mutations in this case increased the sensitivity for the resistance assay from 55.8% to 82.3%, while the assay specificity for KAN resistance remained very high at 99.5% (95% CI, 98.2 to 100%). Thus, based on an MGIT-based susceptibility test, the deei assay allowed the detection of an additional 29 KAN-resistant samples without affecting specificity (Table 4).

Example 5. Relationship between mutations detected by the assay and MIC

The discordance between resistance as defined by the assay disclosed herein and resistance as defined by two phenotypic susceptibility testing methods disclosed herein primarily involved isolates with mutations in the deeis promoter. Previous studies have shown that mutations in the deeis promoter result in relatively low levels of resistance to KAN, whereas mutations in the rrs gene result in high level resistance to AMK, KAN, and CAP (Campbell 2011; Du, Q., et al., 2013, Diagn Microbiol Infect Dis 77:138-142; Georghiou 2012; and Zaunbrecher 2009). An additional finding in the results was the discordance between the results of the LJ-based susceptibility test versus MGIT. An MIC assay was performed to more carefully explore the relationship between the enrrs and deeis promoter mutations, and their differential susceptibility patterns in the LJ and MGIT systems. Samples that were susceptible to both AMK and KAN (and wild-type in both target regions), or chosen to be representative of the most common mutation types in the two target genes (rrsA1401G and eisG(-10)A, C(-14)T, and G(-37)T) were tested by the MYCOTB method for their MIC. Additional isolates known to be wild-type in both test targets were also tested as controls. The AMK MICs of isolates carrying only deeis promoter mutations (without enrrs mutations) were found to range from 0.25 μg/ml to 2 μg/ml, with most samples showing MICs of 0.5 μg/ml to 1 μg/ml (Fig. 3). Only one deeis promoter mutant had an AMK MIC of 4 μg/ml. Control isolates without deeis promoter mutation had MICs in the 0.25 μg/ml to 0.5 μg/ml range. Thus, the AMK MICs of isolates with wild-type and mutant deeis promoter sequences overlapped substantially. In contrast, the KAN MICs for most deeis promoter mutants ranged from 5 μg/ml to 20 μg/ml, with one isolate showing a MIC of 40 μg/ml (Fig. 3). Only two deeis promoter mutants had MICs as low as 2.5 μg/ml. Isolates with wild-type deeis promoter sequences showed MICs between 0.6 μg/ml and 2.5 μg/ml, which is 2- to 30-fold lower than the mean MIC of deeis promoter mutants (Fig. 3). Thus, in contrast to the situation with AMK, the KAN MICs of wild-type isolates had very little overlap with the KAN MICs of deeis promoter mutants. These results strongly suggest that deeis promoter mutants should be considered to have a low to moderate level of resistance to KAN even if resistance is not detected in LJ-based or even MGIT-based susceptibility assays.

Example 6. Specificity of the assay against bacteria other than M.Tb.

The analytical specificity of the assay was tested against a panel of 18 nontuberculous mycobacteria (NTM) species obtained from the ATCC repository (Manassas, VA, USA), 121 clinical NTM strains representing 26 species, and 18 species of Gram-positive and Gram-negative bacteria. The target region in the assay here is highly conserved among different NTM species. Therefore, the assay generated a Tm of 70 °C (which is identical to the Tm generated in the presence of wild-type M. tb DNA) for all NTM tested as expected based on the expected sequence homology to M. xenopi, which did not generate any Tm. NTM species that generate Tm values identical to aminoglycoside-susceptible M. tb would not be expected to elicit a false resistance test result. When M. tb DNA from AMK- and KAN-resistant mutant strains was mixed with a 10- to 20-fold excess of NTM DNA, a distinct two-fold Tm peak was produced by the assay, corresponding to a mutant Tm value of the M. tb target and a wild-type Tm value of the NTM sequence (data not shown) indicating that resistance-associated mutations in M. tb can be detected by the assay in this case even in the presence of a large background of NTM DNA. No visible melting curve was generated by the deei probes in the presence of any NTM species tested even when 10<7> genomic equivalents of DNA were added to the PCR assay. None of the Gram-positive or Gram-negative bacteria produced Tm values for any of the derrso oei SMBs; therefore, they did not cause any false resistance calls to be made by the assay.

Example 7. Additional genetic causes of resistance to AMK and KAN

The study in this example included 22 samples that were resistant to AMK and/or KAN, but had the wild-type gene and deeis promoter sequences. Recent research has suggested that mutations in the 5' untranslated region (UTR) of the whiB7 gene may cause aminoglycoside resistance in M. tb(27). To determine whether mutations in whiB7 might be responsible for some of the phenotypically resistant, but susceptible isolates in the assay, all 22 samples were sequenced in a 412 bp region 5' of the whiB7 gene start site plus a portion of the whiB7 open reading frame. As a control set, 30 randomly chosen generally susceptible isolates were also sequenced. Of the 22 discordant isolates, six isolates from three patients showed mutations in the 5'UTR of whiB7. One sample had a cytosine deletion at position 138 in the 5'UTR, two samples from one patient contained an A to G mutation at position 237, and the remaining three samples from a single patient showed an A to C mutation at position 273 (Table 5) considering the transcription start site as 1 (Reeves, A. Z., 2013, 57:1857-1865). Three samples from a single patient failed to generate any amplification from the 5'UTR after repeated PCR attempts despite working positive PCR controls. This suggested the presence of a large deletion in the 5'UTR region as a 275 bp fragment could be readily amplified from within the whiB7 ORF for all three samples. All samples with mutations in whiB7 were resistant only to KAN, which is consistent with the presumed mechanism of action of whiB7 via upregulation of the is gene (Reeves 2013). The KAN MICs for these isolates were also low at 5 μg/ml, which is similar to that observed for the is promoter mutants. None of the 30 control samples that were susceptible to aminoglycosides had any mutations in the 5'UTR of the whiB7 gene. Further studies are needed to confirm the relationship of these mutations and the deletion in the 5'UTR of the whiB7 gene to aminoglycoside resistance. However, the absence of such mutations in the susceptible strains implies that they may have some role to play in aminoglycoside resistance and future assays could target these mutations to improve the sensitivity to detect low-level KAN resistance.

Table 3: Melting temperature (Tm) values of the rrs and eis probes tested against clinical DNA with wild-type and mutant sequences

SD represents /- the standard deviation of the Tm values for each of the probes for the different clinical samples and dTm represents the difference in Tm of the mutant sequences relative to the wild-type sequences for each probe.

SD; standard deviation, dTM; delta Tm

Probes 1, 2 and 3 correspond to probes rrs-1400, eis-1 and eis-2 respectively.

Table 4: Susceptibility of clinical strains to AMK and KAN by the LJ and MGIT methods and their relationship with the mutations present in the rrs gene and the eis promoter region

LJ and MGIT involve susceptibility testing using the LJ proportions and MGIT methods, respectively.

Table 5: Susceptibility of clinical strains to AMK and KAN and mutations in the 5' untranslated region of the whiB7 gene

LJ and MGIT involve susceptibility testing by the LJ and MGIT proportion methods, respectively.

ND; not determined, NM; no mutation, R; resistant, S; susceptible.

Claims (13)

1. A kit for detecting multidrug-resistant Mycobacterium tuberculosis in a sample, the kit comprising: a first nucleic acid probe for a first region, said first region being a gene knockout of Mycobacterium tuberculosis, wherein the first nucleic acid probe comprises a probe sequence having a sequence of SEQ ID No: 56, and a second nucleic acid probe for a second region selected from the group consisting of genrpoB, gengyrA, gengyrB, deinhA promoter, genrrs, deeis promoter, and genembB of Mycobacterium tuberculosis. 2. The kit of claim 1, wherein the first or second nucleic acid probe is labeled. 3. The kit of claim 2, wherein the first or second nucleic acid probe is labeled with a fluorophore and a quencher at its two ends, respectively. 4. The kit of claim 3, wherein the fluorophore is fluorescein, cyanine 5, TexasRed or TAMRA. 5. The kit of claim 3 or 4, wherein the shield is BHQ1, BHQ2 or DABCYL. 6. The kit of any one of claims 1-5, wherein the first or second nucleic acid probe is a linear probe, a Taqman probe, a molecular beacon probe or a neglected molecular beacon probe. 7. The kit of any one of claims 1-5, further comprising a pair of forward and reverse primers for amplifying a portion of a genekatG of Mycobacterium tuberculosis, wherein the forward primer comprises a sequence that is at least 80% identical to SEQ ID NO: 7, 8 or 9 and the reverse primer comprises a sequence that is at least 80% identical to SEQ ID NO: 10 or 11. 8. The kit of any one of claims 1-7, wherein the second region is the genrpoBo the genrrs. 9. The kit of claim 8, further comprising a DNA polymerase, extension nucleotides and a buffer. 10. A method for detecting multidrug-resistant Mycobacterium tuberculosis in a sample using a kit according to any one of claims 1 to 9. 11. A method for detecting multidrug-resistant Mycobacterium tuberculosis in a sample, comprising: amplifying a first nucleic acid target sequence with a first primer pair to generate a first amplicon, the first primer pair being specific for a portion of a region of the gene katG, wherein the forward primer comprises a sequence that is at least 80% identical to SEQ ID NO: 7, 8 or 9 and the reverse primer comprises a sequence that is at least 80% identical to SEQ ID NO: 10 or 11, detecting a first mutation in the first amplicon, determine the presence of multidrug-resistant Mycobacterium tuberculosis in the sample based on the presence of the first mutation, where the presence of the mutation is indicative of drug resistance; where the detection stage is carried out by a process comprising contacting the first amplicon with a first probe specific for said mutation under conditions conducive to hybridization to form a probe-target hybrid; performing a melting temperature (Tm) analysis to determine an assay Tm value for said probe-target hybrid; and compare the test Tm value with a predetermined reference Tm value, whereby the test Tm value, if different from the default Tm value, indicates the presence of the mutation, wherein the first probe comprises a probe sequence having a sequence of SEQ ID No: 56. 12. The method of claim 11, wherein the test Tm value, if less than the predetermined reference Tm value, indicates the presence of the mutation. 13. The method of any one of claims 11-12, further comprising amplifying a second nucleic acid target sequence with a second primer pair to generate a second amplicon, the second primer pair being specific for a portion of a second region selected from the group consisting of the genrpoB, gengyrA, gengyrB, deinhA promoter, genrrs, deeis promoter, and genembB.